Searching for Novel AntiCRISPRs Using Bioinformatics and Wet-Lab Studies
Tommy W
Marin Academy Research Collaborative Program
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Shift in Project Focus
Shift in Project Focus
In October 2021, I connected with Dr. Blackburn-Marino, a postdoc researcher at UCSF's Bondy-Denomy lab. The lab studies the evolutionary arms race between bacteriophages and bacteria, and Dr. Marion's research focuses on discovering novel anti-CRISPR (Acr) candidates using bioinformatics, then testing the candidates in wet-lab studies to determine whether or not they are anti-CRISPRs. While she has moved on to classifying bacterial immune systems and plans to study the effects on pathogenic bacteria of removing associated prophages, I am joining her lab and taking over the search for novel antiCRIPSRs. To this end, I have mainly been familiarizing myself with NCBI's BLAST, a bioinformatics tool to compare the genomes of different organisms. I am using methods like searching for Acr genes within bacteria that contain self-targeting spacers, and a guilt-by-association method of genomic comparison. I will then move to wet lab studies in Dr. Marino's lab, confirming if the candidates act as antiCRISPRs or antiCRISPR associated (Aca) proteins. The research project outlined below (hyperlink in red button) regarding methods of bacteriophage engineering is an area I planned to study before connecting with Dr. Marino, and hope to return to in the future.
CRISPRs
CRISPRs
CRISPR, or Clustered Regularly Interspaced Palindromic Repeats evolved as a bacterial defense system against infecting bacteriophages. CRISPRs are sequences in a bacterial genome that store nucleotide sequences of bacteriophages that have previously infected the phage. In the event that the same phage attempts to infect the bacteria again, the stored phage DNA in the CRISPR sequence triggers the formation of a CRISPR-associated (Cas) protein. The Cas protein is then guided to the infecting phage genetic material by a sequence of guide RNA (gRNA), and the Cas protein cleaves the phage genes. This prevents the phage from replicating in the bacterium. Recently, CRISPR has been adapted from bacteria to be used as a genetic engineering system. It allows for precise insertions and deletions in gene and has the potential to eradicate hereditary diseases. An issue is that the longer a CRISPR system stays in a cell, the greater the likelihood that it will edit a locus in the genome that it was not intended to. Prior to the discovery of antiCRISPRs, there were very few ways to control CRISPR after it was engineered into a cell, but antiCRISPR research provides a much-needed control mechanism.
antiCRISPRs
antiCRISPRs
AntiCRISPRs are proteins that inhibit normal CRISPR function. They evolved in bacteriophages as a defense against bacterial CRISPR, an immune system that prevented the proliferation of phages in their hosts. The co-evolution of CRISPRs and antiCRISPRs is referred to as an evolutionary arms race. AntiCRISPRs can be used to end a gene therapy treatment or to help guide treatments to the right part of the body. AntiCRISPRs are specific in the CRISPR system that they target, and have a variety of mechanisms for disabling a CRISPR system. Some of these include the antiCRISPR protein binding to a Cas protein and preventing it from cleaving the DNA. Another mechanism that antiCRISPRs employ involves the prevention of Cas transcription from a bacterial genome. This makes it so a cell is not able to synthesize the necessary proteins for the formation of the Cas protien.
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